T. A. Zargarova

Synthetic β-endorphin-like peptide immunorphin binds to non-opioid receptors for β-endorphin on T lymphocytes

Abstract The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364–373 of the CH3 domain of human immunoglobulin G heavy chain was found to compete with [125I]β-endorphin for high-affinity receptors on T lymphocytes from the blood of healthy donors (Ki = 0.6 nM). Besides immunorphin, its synthetic fragments H-Val-Lys-Gly-Phe-Tyr-OH (Ki = 15 nM), H-Leu-Val-Lys-Gly-Phe-Tyr-OH (Ki = 8.0 nM), H-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (Ki = 3.4 nM), H-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (Ki = 2.2 nM), H-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (Ki = 1.0 nM) possessed the ability to inhibit specific binding of [125I]β-endorphin to T lymphocytes. Tests of the specificity of the receptors revealed that they are not sensitive to naloxone and Met-enkephalin, i.e. they are not opioid receptors. Kd values characterizing the specific binding of 125I- labeled immunorphin and its fragment H-Val-Lys-Gly-Phe-Tyr-OH to the receptors have been determined to be 7.4 nM and 36.3 nM, respectively.

Macrophage-stimulating peptides VKGFY and cyclo(VKGFY) act through nonopioid β-endorphin receptors☆

We have synthesized two peptides, VKGFY and cyclo(VKGFY) (referred to as pentarphin (PNT) and cyclopentarphin (cPNT), respectively), and found that both peptides at 1 nM concentration increased the adhesion and spreading of murine peritoneal macrophages as well as their bactericidal activity in vitro, as shown by phagocytosis of Salmonella typhimurium virulent strain 415. PNT administered intraperitoneally at dose 20 microg/mouse on day 7, 3, and 1 prior to the isolation of macrophages also enhanced the macrophage adhesion and spreading. The receptor binding characteristics of PNT and cPNT were examined using 125I-labeled PNT. The binding of labeled PNT to peritoneal macrophages was high-affinity (K(d)=3.6 nM) and saturable. It was not inhibited by naloxone (NAL) or [Met(5)]enkephalin ([Met(5)]ENK) but completely inhibited by unlabeled cPNT (K(i)=2.6 nM), immunorphin (IMN, decapeptide SLTCLVKGFY, corresponding to the IgG heavy-chain sequence 364-373) (K(i)=3.2 nM) or beta-endorphin (beta-END) (K(i)=2.8 nM). Thus, the effects of PNT and cPNT on macrophages are mediated by NAL-insensitive receptors common for PNT, cPNT, IMN, and beta-END.

Effect of Synthetic β-Endorphin-Like Peptide Immunorphin on Human T Lymphocytes

Abstractβ-Endorphin and the synthetic β-endorphin-like decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (referred to as immunorphin), corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain, were shown to stimulate concanavalin A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met5]Enkephalin and the antagonist of opioid receptors naloxone examined in parallel were inactive. The stimulating effect of β-endorphin and immunorphin on T lymphocyte proliferation is not inhibited by naloxone. Studies on receptor binding of 125I-labeled immunorphin to T lymphocytes revealed that it binds with high affinity to naloxone-insensitive receptors (Kd = 7.0 ± 0.3 nM)). Unlabeled immunorphin completely inhibits 125I-labeled β-endorphin specific binding to naloxone insensitive receptors on T lymphocytes (Ki = 0.6 ± 0.1 nM)). Thus, β-endorphin and immunorphin interact with common naloxone insensitive receptors on T lymphocytes.

Synthetic peptide SLTCLVKGFY competes with β-endorphin for naloxone-insensitive binding sites on rat brain membranes

The synthetic decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain and its synthetic fragment VKGFY were found to compete with 125I-labeled beta-endorphin for high-affinity naloxone-insensitive binding sites on membranes isolated from the rat brain cortex (K(i)=1.18+/-0.09 and 1.58+/-0.11 nM, respectively). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to [Met(5)]enkephalin and [Leu(5)]enkephalin as well. The K(d) values characterizing the specific binding of 125I-labeled immunorphin and its fragment Val-Lys-Gly-Phe-Tyr to these binding sites were determined to be 2.93+/-0.27 nM and 3.17+/-0.29 nM, respectively.

Synthetic Peptide VKGFY and Its Cyclic Analog Stimulate Macrophage Bactericidal Activity through Non-Opioid β-Endorphin Receptors

We synthesized linear and cyclic pentapeptides corresponding to the sequence 369-373 of human immunoglobulin G heavy chain—VKGFY (referred to as pentarphin and cyclopentarphin, respectively). The effect of pentarphin and cyclopentarphin on phagocytosis of Salmonella typhimurium virulent 415 strain bacteria by mouse peritoneal macrophages in vitro was studied. Control experiments showed that macrophages actively captured these bacteria, but did not digest them: the captured microbes were viable and continued to proliferate inside the phagocytes; within 12 h all macrophage monolayer was destroyed (incomplete phagocytosis). If 1 nM pentarphin or cyclopentarphin was added to the cultivation medium, macrophage bactericidal activity was significantly increased and they digested all captured microorganisms within 6 h (complete phagocytosis). To study the receptor binding properties of pentarphin and cyclopentarphin we prepared 125I-labeled pentarphin (179 Ci/mmol specific activity). The binding of 125I-labeled pentarphin to mouse peritoneal macrophages was highaffinity (Kd = 3.6 ± 0.3 nM) and saturable. Studies on binding specificity revealed that this binding was insensitive to naloxone and [Met5]enkephalin, but completely inhibited by unlabeled cyclopentarphin (Ki = 2.6 ± 0.3 nM), immunorphin (Ki = 3.2 ± 0.3 nM), and β-endorphin (Ki = 2.8 ± 0.2 nM). Thus, the effects of pentarphin and cyclopentarphin on macrophages are mediated by naloxone-insensitive receptors common for pentarphin, cyclopentarphin, immunorphin, and β-endorphin.

The Interaction of Synthetic Decapeptide SLTCLVKGFY with Human T Lymphocytes

The synthetic peptide SLTCLVKGFY, corresponding to the 364–373 amino acid sequence of the human IgG heavy chain (Immunorphin), was found to compete with [125I] β-endorphin for binding by high-affinity receptors on T lymphocytes isolated from the blood of healthy donors (Ki0.6 nM). The fragments 3–10, 4–10, 5–10, and 6–10 of Immunorphin also inhibited the binding (Ki2.2, 3.4, 8.0, and 15 nM, respectively). Specificity of these receptors was studied: they turned out to be insensitive to naloxone and [Met]enkephaline and, therefore, are not opioid. The Kdvalues of the specific binding of 125I-labeled Immunorphin and its 6–10 fragment to the receptor were found to be 7.4 and 36.3 nM, respectively.

[An ACTH-like peptide immunocortin: effect on the activity of cells from the rat adrenal cortex].

The effect of immunocortin, an ACTH-like decapeptide VKKPGSSVKV corresponding to the 11–20 sequence of the variable part of the human IgG1 heavy chain on the content of 11-hydroxycorticosteroids (CS) in rat adrenal glands and blood serum in vivo was studied. An intramuscular injection of immunocortin at a dose of 10 μg/kg was found in an hour to induce a twofold decrease in CS content in the adrenal glands and a 1.8-fold increase in the blood serum CS content. At the same time, an immunocortin dose of 100 μg/kg exerted practically no effect on the CS content and its dose of 1000 μg/kg increased the CS content both in adrenal glands and in blood serum by 1.6 and 2.2 times, respectively. Four hours after the injection of any of the three doses of immunocortin, the CS content in adrenal glands did not differ from the control value, and after 24 h the content decreased threefold. Immunocortin was shown to be bound by the ACTH receptors in the membranes of the rat adrenal cortex with a high affinity and specificity (inhibiting the specific binding of 125I-labeled ACTH-(11–24) peptide with Ki of 1.2 nM).